WO2014129773A1 - Method for preparing polymer microparticles by spray process - Google Patents

Abstract

The present invention relates to a method for preparing polymer microparticles by a spray process, wherein a polymer solution obtained by dissolving a polyester-based polymer in ethylene carbonate (hereinafter, referred to as "EC"), which is a solvent, is sprayed at a low temperature hydrocarbon or alcohol solution, thereby preparing frozen EC/polymer microparticles, the frozen EC/polymer microparticles are dissolved in a salt aqueous solution, thereby dissolving and removing EC, and the residual EC in water is removed by washing.

Description

Manufacturing method according to spray process of polymer fine particles

The present invention relates to a manufacturing method according to the spray process of the polymer microparticles. More specifically, a polymer solution obtained by dissolving a polyester polymer in ethylene carbonate (hereinafter referred to as "EC") as a solvent is sprayed onto a low temperature hydrocarbon or alcohol solution to produce frozen EC / polymer microparticles. It relates to a method for preparing polymer microparticles by dissolving and removing EC in water.

Porous biodegradable polymer scaffolds are widely used as templates for various tissue regeneration. The support requires a porous structure with good interpore connectivity to promote nutrition and oxygen supply for sufficient cell adhesion density, cell proliferation and differentiation.

There are various methods of preparing a porous biodegradable polymer support, among which porogen leaching is most widely used. Pore-forming particles are a variety of particles, such as salts, effervescent salts, carbohydrates, hydrocarbon waxes, and is a method of forming pores by selectively dissolving or foaming the pore-forming particles in the polymer / solvent / pore-forming particle mixture. Other methods include emulsification / freeze drying, phase separation, expansion of critical liquid phases, and three-dimensional inkjet printing. (AG Mikos, G. Sarakinos, SM Leite, JP Vacanti, R. Langer, Biomaterials, 14) 1993) 323-330; Z. Ma, C. Gao, Y. Gong, J. Biomed.Mater.Res. 67B (2003) 610-617; A. Park, B. Wu, LG Griffith, J. Biomater.Sci Polym.Ed. 9 (1998) 89-110).

The porous polymer support may be useful for bone, cartilage and liver regeneration by inducing cell adhesion and differentiation. However, these supports are implanted into the body through surgical operations, which is a physical and economic burden on the patient. Therefore, a method of injecting a biodegradable polymer support by injection is developed to minimize patient inconvenience. This is a method of forming a hydrogel by injection of a polymer solution containing cells and then photo-crosslinking or sol-gel development (J. j. Marler, A. Guha, J. Rowley, R. Koka, D. Monney, J. Upton, J. p. Vacanti, Plast.Reconstr. Surg. 105 (2000) 2049-2058; S. He, MJ Yaszemski, AW Yasko, PS Engel, AG Mikos, Biomaterials, 21 (2000) 2389-2394) .

However, these hydrogels do not provide an ideal environment for cells that need to adhere to a solid surface, and their mechanical strength is weak, making it difficult to protect the cells enclosed therein. In order to solve this drawback, a wide range of natural and synthetic microparticles, including Cultispher, a microparticle made of gelatin with a porous structure, have been used in animal cell culture, but they have poor biocompatibility and unsatisfactory mechanical strength. There are disadvantages.

Currently used microparticles for injection is the Emulsification-Solvent Evaporation Method. Among them, the W / O / W dual emulsification method undergoes two emulsification steps, and the porous structure is determined according to the stability of the first emulsification step, W / O emulsion. Since emulsion is thermodynamically unstable, it is difficult to manufacture because the aqueous phase and the organic phase are separated from each other through a process such as coalescence, fusion, and phase separation (M. Kanouni, HL Rosano). , N. Naouli, Adv.Colloid Interface Sci. 99 (2002) 229-254; AJ Webster, ME Cates, Langmuir, 14 (1998) 2068-2079).

In addition, fine particles including a W / O / W double emulsification step of forming a W / O emulsion by adding an aqueous solution in which an effervescent salt is dissolved in an organic phase in which an aliphatic polyester polymer is dissolved, and redispersing and emulsifying it in an aqueous solution containing a hydrophilic surfactant. There is a method for preparing a carrier (see Korean Patent No. 801194). The particulate carrier has properties such as biodegradability, high porosity, excellent interconnectivity between pores, but has a weak mechanical strength and a difficult mass production process.

Recently developed biodegradable polymer microparticle manufacturing method is to dissolve the biodegradable polymer in DMSO (Dimethyl Sulfoxide) and spray it in a low temperature hydrocarbon solution to freeze the DMSO / polymer solution and then remove the DMSO from the low temperature salt solution It is a method for producing biodegradable polymer microparticles (see Korean Patent No. 1105292). Thus prepared microparticles have a high porosity, excellent mechanical strength, excellent cell affinity, but has the disadvantage of using only hydrocarbon as a freezing solvent in the injection process, the most important process of the manufacturing process. Hydrocarbons have a low flash point and have a high risk of fire, which makes them difficult to use in mass production. Therefore, development of a manufacturing process that can solve these problems is required.

Therefore, there has been a demand for development of a low-risk, environmentally friendly mass production process for polymer microparticles having excellent biocompatibility, biodegradability, porosity, mechanical strength, cell affinity, and the like.

Accordingly, the present inventors have studied to develop a method for overcoming the technical disadvantages of the existing microparticle manufacturing process, and the polymer / EC solution made by dissolving a biodegradable polyester polymer in ethylene carbonate (EC). Because of its high melting point (MP, 37 ° C), it is possible to freeze polymers / EC solutions not only in low temperature hydrocarbon solutions but also in alcoholic solutions, so that EC is dissolved in water, resulting in excellent biocompatibility, biodegradability and porous polymer microparticles. It was found that can be prepared, to complete the present invention.

In order to achieve the above object,

The biodegradable polyester polymer is dissolved in ethylene carbonate (EC), and the obtained polymer / EC solution is a hydrocarbon solution or alcohol (methanol) having a carbon number of 5 to 10 (C ₅ to C ₁₀ ) of -20 ° C to 0 ° C. , Ethanol) solution to obtain a frozen polymer / EC microparticles, and put it in an aqueous salt solution to dissolve and remove EC, and wash and remove the remaining EC with water to prepare a biodegradable polymer microparticles.

Hereinafter, the present invention will be described in detail.

First, an ethylene carbonate (EC) solution of a biodegradable polyester polymer is described.

In the present invention, the biodegradable polyester-based polymer is an aliphatic polyester-based polymer, but is not limited thereto. Polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactic acid-co Poly (D, L-lactic-co-glycolic acid, PLGA), polycaprolactone (PCL), poly (valerolactone), poly (hydroxybutyrate), poly (hydroxy valerate) Or derivatives thereof, and may be a single or a mixture of two or more components. Preferably PLA, PGA, PLGA, PCL or mixtures thereof, more preferably PLA, PLGA or PCL. These polymers preferably have an average molecular weight of 10,000 to 250,000. However, the method for preparing biodegradable polymer microparticles of the present invention has the characteristics of easily producing spherical microparticles and of easily controlling the size of microparticles. It is not limited.

Biodegradable polyester-based polymer solution in the present invention can be used by variously controlled by dissolving the polymer in EC (Ethylene carbonate) so as to have a concentration of 1% -25% (w / v), prepared through such a concentration control The porosity of the biodegradable porous microparticles can be controlled. When the concentration of the polymer solution is less than 1%, the mechanical strength of the microparticles is weak, which makes them less practical. When the concentration of the polymer solution is more than 25%, the viscosity is too high.

In the present invention, the organic solvent used to dissolve the aliphatic polyester polymer has a high freezing point (Melting Point 37 ° C.) and uses EC (Ethylene carbonate) which is well mixed with water.

In the present invention, the hydrocarbon solution is a hydrocarbon having 5 to 10 (C ₅ to C ₁₀ ) carbon atoms, for example, Pentane, Hexane, Heptane, which are phase-separated from EC without freezing below zero degrees Celsius. , Saturated hydrocarbons such as octane, nonane, decane, petroleum ether, and mixtures thereof, preferably using volatile n-hexane. good. Due to the high volatility of n-hexane, it can finally be easily removed during drying. Hydrocarbons with less than 5 carbon atoms are too volatile and difficult to manufacture, and hydrocarbons having 10 or more carbon atoms are not practical. The temperature of the hydrocarbon solution is preferably below the melting point of the EC for freezing of the EC. More preferably, it is -20 ° C to 0 ° C, most preferably -10 ° C to -5 ° C to facilitate freezing of EC and formation of fine particles.

In the present invention, the alcohol solution is an alcohol having 1 to 2 carbon atoms (C ₁ to C ₂ ), for example, alcohols such as methanol, ethanol, and aqueous solutions thereof, preferably ethanol which is environmentally friendly and nontoxic. And its aqueous solution are preferred. When the polymer / EC solution is sprayed on a low temperature alcohol solution, some EC slowly melts in the alcohol solution, and thus the polymer / EC frozen microparticles are formed. Therefore, alcohols having 3 or more carbon atoms cannot dissolve the polymer.

The temperature of the alcohol solution is preferably below the melting point of the EC for freezing of the EC. More preferably, it is -20 ° C to 0 ° C, most preferably -10 ° C to -5 ° C to facilitate freezing of EC and formation of fine particles.

Frozen polymer / EC microparticles prepared from low temperature hydrocarbons are added to an aqueous salt solution to dissolve and remove EC and wash with water to obtain polyester polymer microparticles of the present invention.

Frozen polymer / EC microparticles prepared in a low temperature alcohol solution are washed with distilled water to obtain the polyester-based polymer microparticles of the present invention.

In the present invention, it is preferable that the aqueous salt solution is kept in a freezing state at 0 ° C or less. In order to ensure the stability of the microparticles during the preparation, it is preferable to remove the EC with a solution of 0 ° C. or less. Therefore, it is preferable to use an aqueous NaCl or CaCl ₂ solution at a concentration of 5% to 30%. Preferably, the aqueous salt solution may use a 20% to 25% sodium chloride (NaCl) solution, it is preferable to use from -20 ℃ to 0 ℃. Removal of the salts after removal of the EC with aqueous salt solution can be carried out by washing with excess water, preferably deionized distilled water (DDW) to remove residual EC and salts.

Biodegradable polymer microparticles can be produced by such a method. The biodegradable polymer microparticles prepared by the present invention had a yield of 80% or more and had a diameter of 30 μm to 1,000 μm. The diameter of the biodegradable polymer microparticles of the present invention can be adjusted according to the injection amount and the amount of injection air during the injection of the EC polymer solution, the porosity of the biodegradable polymer microparticles can be controlled by the polymer concentration of the EC polymer solution. .

Therefore, the present invention is excellent in biocompatibility, biodegradability, porosity, etc. so that it can be used as a cell carrier and cell culture medium, and can be injected into a syringe to be used as a cell carrier for tissue regeneration to restore damaged tissues by injecting in vivo. It provides a method for mass production of biodegradable polymer microparticles.

Figure 1 is a photograph of the microparticles prepared according to the method of the present invention by electron microscopy. (X 700; polymer solution concentration: 5%; injection amount: 1 ml / min; air injection rate: 5 L / min; freezing Solvent: n-Hexane; Freezer Temperature: -10 ℃)

Figure 2 is a photograph observing the microparticles prepared according to the method of the present invention with an electron microscope. (X 1,000; polymer solution concentration: 5%; injection amount: 1 ml / min; air injection rate: 5 L / min; freezing Solvent: n-Hexane; Freezer Temperature: 0 ℃)

Figure 3 is a photograph observing the microparticles prepared according to the method of the present invention with an electron microscope. (X 1,500; polymer solution concentration: 5%; injection amount: 1 ml / min; air injection rate: 5 L / min; freezing Solvent: EtOH; freezer temperature: -10 ° C)

Figure 4 is a photograph observing the microparticles prepared according to the method of the present invention with an electron microscope. (X 500; polymer solution concentration: 5%; injection amount: 1 ml / min; air injection rate: 5 L / min; freezing Solvent: EtOH; freezer temperature: -5 ° C)

5 is a photograph observing the microparticles prepared according to the method of the present invention by an electron microscope. (X 700; polymer solution concentration: 5%; injection amount: 1 ml / min; air injection rate: 5 L / min; freezing Solvent: 75% aqueous solution of EtOH; freezer temperature: -5 ° C)

Figure 6 is a photograph observing the microparticles prepared according to the method of the present invention with an electron microscope. (X 800; polymer solution concentration: 5%; injection amount: 1 ml / min; air injection rate: 5 L / min; freezing Solvent: 75% aqueous solution of EtOH; freezer temperature: 5 ° C.)

Of the examples described below, example 1 is the best mode.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Examples 1-6

Example 1 . 2.5 g of polylactic acid (PLA) having an average molecular weight of 110,000 was dissolved in 50 ml of EC (ethylene carbonate) to prepare a 5% (W / V) polymer solution. The polymer solution was injected into n-hexane cooled to −10 ° C. under the conditions of 1 ml / min injection amount and 5 L / min amount of injection air. At this time, the injected polymer solution is frozen in spherical form at low temperature n-hexane.

Obtaining the frozen microparticles to dissolve the EC component in 100 ml of 25% (w / v) NaCl aqueous solution cooled to -10 ℃ for 48 hours, and then filtered to obtain the polymer microparticles from which EC is removed. This was again washed with 500 ml distilled water to remove residual EC and then lyophilized to complete the biodegradable polymer microparticles of the present invention.

Example 2 . In addition, the biodegradable polymer microparticles were prepared in the same manner as in the above method 1 except that the polymer solution dissolved in EC was dropped in -5 ° C. and n-hexane.

Example 3 . 2.5 g of polylactic acid (PLA) having an average molecular weight of 110,000 was dissolved in 50 ml of EC (ethylene carbonate) to prepare a 5% (W / V) polymer solution. The polymer solution was injected into ethanol (Ethanol) cooled to −10 ° C. under the conditions of 1 ml / min injection amount and 5 L / min amount of injection air. At this time, the injected polymer solution is frozen in a round shape in low temperature ethanol.

Obtained frozen microparticles were washed with 500 ml distilled water to remove residual EC and then lyophilized to complete the preparation of the biodegradable polymer microparticles of the present invention.

Example 4 . In addition, the biodegradable polymer microparticles were prepared in the same manner as in the above method 3 except that the polymer solution dissolved in EC was dropped in -5 ° C ethanol.

Example 5 . In addition, the biodegradable polymer microparticles were prepared in the same manner as in the above method 3 except that the polymer solution dissolved in EC was dropped in 75% ethanol aqueous solution at -10 ° C.

Example 6 In addition, the biodegradable polymer microparticles were prepared in the same manner as in the above method 3 except that the polymer solution dissolved in EC was dropped in an aqueous solution of 75% ethanol at 5 ° C.

Experimental Example

In order to confirm the properties of the biodegradable polymer microparticles prepared in Examples 1 to 6, the shape and production yield of the microparticles were measured. At this time, the shape of the microparticles was measured by taking an electron microscope photograph, and the production yield was calculated by measuring the amount of the finally obtained microparticles relative to the amount of the injected polymer.

As a result, as shown in Figs. 1 to 6 and Table 1, spherical microparticles were prepared in the frozen solvent of the EC polymer solution in n-hexane, and the rounded solvent was prepared in the ethanol and its aqueous solution. The shape of the microparticles prepared according to the method of the present invention was a size suitable for administration in the body, and in particular, the microparticles prepared in ethanol and its aqueous solution were prepared in a round-shaped cell to which cells are more adhered to, and thus the cell transporter for tissue regeneration. It was judged that it could be used more usefully.

Table 1

Manufacturing condition; 5% PLA / EC solution, spray rate; 1 ml / min, air injection amount; 5 L / min		Microparticle morphology	Fine particle size μm	yield%
Frozen Solvent	Refrigeration temperature, ℃
n-Hexane	-10	Porosity	100-300 μm	81
	0			80
EtOH	-10	Round		83
	-5			82
75% EtOH	-5			82
	5			81

It can be seen that the present invention is a useful invention for providing a method for preparing polymer microparticles which can inject spherical and round-shaped biodegradable polymer particulate carriers by syringe. Particularly, the round-shaped microparticles are expected to improve cell adhesion, which is expected to contribute to the bio industry through their use as cell carriers.

Claims (5)

1. The biodegradable polyester polymer is dissolved in ethylene carbonate (EC), and the obtained polymer / EC solution is a hydrocarbon solution of 5 to 10 carbon atoms (C ₅ to C ₁₀ ) or methanol or ethanol at -20 ° C to 0 ° C. Method of preparing a biodegradable polymer microparticles by spraying the alcohol solution selected from among to obtain a frozen polymer / EC microparticles, put it in a salt aqueous solution to dissolve and remove EC, and wash and remove the residual EC with water.
2. The method of claim 1, wherein the biodegradable polyester-based polymer is polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L- lactic acid-co-glycolic acid) (Poly (D, L-lactic-co- biodegradable polymer having a weight average molecular weight of 10,000 to 250,000 selected from glycolic acid, PLGA), polycaprolactone (PCL), poly (valerolactone), poly (hydroxybutyrate) and poly (hydroxy valerate) Method for producing polymer microparticles.
3. The method for producing biodegradable polymer microparticles according to claim 1, wherein the biodegradable polyester polymer is dissolved in ethylene carbonate (EC) so as to be 1 to 25% (w / v).
4. According to claim 1, wherein the hydrocarbon (hydrocarbon) is made of pentane (hexane), hexane (hexane), heptane (heptane), octane, nonane (nonane), decane (decane) and petroleum ether (petroleum ether) Method for producing biodegradable polymer microparticles selected from the group.
5. The method of claim 1, wherein the aqueous salt solution is 5 to 30% (w / v) of NaCl or CaCl ₂ aqueous solution.

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